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Tracking Light-Induced Electron Transfer towards O2 ina Hybrid Photoredox-Laccase System

Rajaa Farran, Yasmina Mekmouche, Nhat Tam Vo, Christian Herrero,Annamaria Quaranta, Marie Sircoglou, Frédéric Banse, Pierre

Rousselot-Pailley, A. Jalila Simaan, Ally Aukauloo, et al.

To cite this version:Rajaa Farran, Yasmina Mekmouche, Nhat Tam Vo, Christian Herrero, Annamaria Quaranta, etal.. Tracking Light-Induced Electron Transfer towards O2 in a Hybrid Photoredox-Laccase System.iScience, Elsevier, 2021, pp.102378. �10.1016/j.isci.2021.102378�. �hal-03190584�

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iScience

Article

Tracking light-induced electron transfer toward O2

in a hybrid photoredox-laccase system

Rajaa Farran,

Yasmina

Mekmouche, Nhat

Tam Vo, ..., Ally

Aukauloo, Thierry

Tron, Winfried

Leibl

winfried.leibl@cea.fr

HighlightsAn electron relay boosts

photoreduction of laccase

Superoxide is efficiently

captured by laccase

preventing formation of

H2O2

Light activation reveals

information on elementary

steps inside the enzyme

Laccase enables O2 as

terminal electron acceptor

for oxidative

photocatalysis

Farran et al., iScience 24,102378April 23, 2021 ª 2021 TheAuthor(s).

https://doi.org/10.1016/

j.isci.2021.102378

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Article

Tracking light-induced electron transfertoward O2 in a hybridphotoredox-laccase system

Rajaa Farran,1,2 Yasmina Mekmouche,3 Nhat Tam Vo,4 Christian Herrero,4 Annamaria Quaranta,1

Marie Sircoglou,4 Frederic Banse,4 Pierre Rousselot-Pailley,3 A. Jalila Simaan,3 Ally Aukauloo,1,4 Thierry Tron,3

and Winfried Leibl1,5,*

SUMMARY

Photobiocatalysis uses light to perform specific chemical transformations in a se-lective and efficient way. The intention is to couple a photoredox cycle with anenzyme performing multielectronic catalytic activities. Laccase, a robust multi-copper oxidase, can be envisioned to use dioxygen as a clean electron sinkwhen coupled to an oxidation photocatalyst. Here, we provide a detailed studyof the coupling of a [Ru(bpy)3]

2+ photosensitizer to laccase. We demonstratethat efficient laccase reduction requires an electron relay like methyl viologen.In the presence of dioxygen, electrons transiently stored in superoxide ions arescavenged by laccase to form water instead of H2O2. The net result is the photoaccumulation of highly oxidizing [Ru(bpy)3]

3+. This study provides ground for theuse of laccase in tandemwith a light-driven oxidative process and O2 as one-elec-tron transfer relay and as four-electron substrate to be a sustainable final electronacceptor in a photocatalytic process.

1Universite Paris-Saclay, CEA,CNRS, Institute forIntegrative Biology of the Cell(I2BC), 91191 Gif-sur-Yvette,France

2Lebanese InternationalUniversity, 146404 Mazraa,Beirut, Lebanon

3Aix Marseille Universite,Centrale Marseille, CNRS,iSm2 UMR 7313, 13397Marseille, France

4Universite Paris-Saclay,Institut de ChimieMoleculaire et des Materiauxd’Orsay (ICMMO), 91405Orsay, France

5Lead contact

*Correspondence:winfried.leibl@cea.fr

https://doi.org/10.1016/j.isci.2021.102378

INTRODUCTION

Electron transfer (ET) reactions are of fundamental importance in biological processes such as photosyn-

thesis and respiration (Winkler, 2005). Understanding how electrons are transported in these complex

systems may help chemists discover new tools to perform sustainable multielectronic chemical transforma-

tions. Currently, chemists and biologists are gathering their efforts to use light energy to drive enzymes to

realize both chemical oxidation and reduction reactions of high importance for our societies (Lee et al.,

2018; Litman et al., 2018; Guo et al., 2018; Schmermund et al., 2019). These targets are gaining much atten-

tion from both a fundamental and applied point of view due to the fact that to date we do not have catalytic

systems that can match the activities and specificities of the biological catalysts. Hence, discoveries in this

field may help develop sustainable chemical processes and also help in the design of more simple and

robust photocatalytic systems (Pellegrin and Odobel, 2011).

Laccases are robust enzymes used in many industrial and biotechnological processes (Mate and Alcalde,

2017; Couto and Herrera, 2006; Agrawal et al., 2018; Riva, 2006). In nature, laccases couple the oxidation

of organic substrates such as phenol derivatives, to the reduction of O2 to water (Madhavi and Lele,

2009; Tron, 2013). The catalytic mechanism of laccases has been extensively studied (Solomon et al.,

2008; Jones and Solomon, 2015; Heppner et al., 2014). At a type 1 (T1) Cu ion site the one-electron oxida-

tion of substrate molecules occurs, and electrons are sequentially transferred inside the protein to a trinu-

clear copper catalytic center (TNC) where O2 is eventually reduced in a four-electron, four-proton reaction

(Jones and Solomon, 2015; Bertrand et al., 2002). The TNC consists of a pair of coupled type 3 (T3) Cu ions

and a mononuclear type 2 (T2) Cu ion (Solomon et al., 1996; Hakulinen and Rouvinen, 2015). Although lac-

cases are active on a wide range of natural substrates including phenol, polyphenols, hydroxyindoles, and

benzenethiols among others, their oxidative power is locked by the one-electron oxidation process taking

place at the T1 site whose potential ranges from 420 to 790 mV versus NHE, thus limiting the scope for the

oxidation of organic substrates. Inspired by this biological tandem of oxidative and reductive processes

performed by MCOs we have been interested in demonstrating that we can employ O2 as the final oxidant

by coupling a photoredox cycle with a laccase. Light-induced ET from the excited state of a photosensitizer

iScience 24, 102378, April 23, 2021 ª 2021 The Author(s).This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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to laccase leads to the reduced enzyme and the generation of a highly oxidizing chromophore. In doing so,

we can use the reductive facet of this family of enzymes as a substitute for the problematic and unsustain-

able sacrificial electron scavengers such as persulfate or cobalt(III) ions widely used in photocatalytic multi-

electronic oxidative processes (Herrero et al., 2011). From our first studies, it appears that the effective

quantum yield for the reduction of the MCO using ruthenium(II) polypyridine-type chromophores in the

presence of a sacrificial electron donor does not exceed 1% (Simaan et al., 2011). When a porphyrin-based

chromophore is used, this yield approaches 35% (Lazarides et al., 2013) but suffers from an intrinsic low sta-

bility of the chromophore in the presence of O2. The poor efficiency observed with the ruthenium photo-

sensitizers was initially conferred to the shorter excited state lifetime of [Ru(bpy)3]2+ (600 ns) as

compared with the zinc porphyrin triplet state (720 ms). However, other factors such as energy transfer

quenching and back electron transfer might also hamper this bimolecular light-activation process.

Such pathways can hardly be assessed from measurements of overall yields under continuous illumination.

To improve the efficiency of the light-driven activation of laccase by [Ru(bpy)3]2+, we set to reinvestigate

the photophysical events occurring upon excitation of a [Ru(bpy)3]2+ chromophore and its

interaction with laccase. In the present study we show that the efficiency of light-induced electron transfer

between our synthetic photosensitizer and the laccase enzyme can be strongly enhanced, from 1% to

20%, by the introduction of methyl viologen (MV2+) as an electron relay. We track the role of methyl violo-

gen acting as an efficient oxidative quencher of the photosensitizer excited triplet state to form a

methyl viologen radical, MV,+, which efficiently reduces the laccase. We furthermore delineate the reaction

pathways under aerobic conditions, where MV,+ reacts with O2 to form superoxide radicals that are effi-

ciently scavenged by the enzyme to perform the 4-electron reduction of O2 to H2O. As a result of these

processes the powerful and long-lived oxidant [Ru(bpy)3]3+ with an oxidizing power of ca. 1300 mV versus

NHE is generated. At all stages experimental data are confronted with kinetic simulations to arrive at a

consistent description of the reaction sequence and the interactions at play between the different

components.

RESULTS AND DISCUSSION

Laccase photoreduction by [Ru(bpy)3]2+*: Marcus versus Forster

We first reinvestigated the interaction between the [Ru(bpy)3]2+ photosensitizer and the laccase enzyme.

Steady-state and time-resolved emission and absorption studies were performed on solutions containing

[Ru(bpy)3]2+ and various concentrations of laccase (LAC3 from Trametes sp. C30) in de-aerated Britton-

Robinson (B&R) buffer at pH 6. As illustrated in Figure S1 in the supplemental information, the

[Ru(bpy)3]2+ chromophore is characterized by its 450-nm metal-to-ligand charge-transfer (MLCT) band

(ε = 14,600 M�1 cm�1) (Kalyanasundaram, 1982a) with an emission band of its excited state at 610 nm,

whereas the oxidized copper(II) ion at the T1 site of the laccase displays an absorption band at

610 nm (ε = 5600 M�1 cm�1) (Solomon et al., 2004). These spectroscopic probes were used accordingly

to monitor the excited state of the photosensitizer and LAC3 photoreduction.

Time-resolved emissionmeasurements with increasing LAC3 concentrations indicate a dynamic quenching

process of the ruthenium excited state by laccase with a diffusion-limited bimolecular rate constant of 6.1

109$M�1s�1 (Figure S2). This emission quenching could be due to energy and/or electron transfer from the

sensitizer to the laccase. If all the quenching were due to ET a quantum yield of 12% of laccase reduction

(compared with the concentration of excited state formed by the laser excitation) would be expected for a

concentration of laccase of 37 mM. However, the amplitude of the measured absorption changes at long

times, i.e., after decay of the excited state (Figure 1, inset) indicates a yield for the laser-flash-induced for-

mation of the charge-separated state (CSS) of [Ru(bpy)3]3+ and reduced LAC3 (CuI) of only 0.7% (see Fig-

ure 1 for details). This yield is 17 times lower than what is expected for the products of the quenching pro-

cess, suggesting that less than 10% of the quenching reactions lead to an effective reduction of laccase

despite the high driving force for the ET from the ruthenium to the copper center T1 of –DG = 1.52 eV esti-

mated from the redox potentials (E(RuIII/RuII*) = �0.84 V; E(CuII/CuI) = 0.68 V) (Kalyanasundaram, 1982a;

Balland et al., 2008; Bock et al., 1979). These findings suggest that the major part of quenching is related

to a Forster-type energy transfer process that can be expected to be rather efficient due to the strong spec-

tral overlap between the [Ru(bpy)3]2+* emission spectrum and the absorption band of the CuII of the T1 site

(Figure S1) (Winkler, 2013). The Forster radius (R0 = 2.6 nm) calculated for this interaction implies that upon

encounter, through-space resonance energy transfer will largely outcompete ET, the rate of which is seven

orders of magnitude slower at that distance (see supplemental information, transparent methods Section

and Figure S5).

2 iScience 24, 102378, April 23, 2021

Figure 1. Laccase photoreduction by [Ru(bpy)3]2+*

Transient absorption kinetics of a solution of [Ru(bpy)3]2+ (15 mM) and LAC3 (37 mM) in argon-purged B&R buffer, pH = 6

after laser flash excitation at 455 nm. Inset: vertical zoom of the data. The absorption transient at 450 nm (red trace)

indicates formation of 9.7 mM of [Ru(bpy)3]2+* by the nanosecond laser flash decaying to a residual long-lived negative

absorption difference of 0.8 3 10�3 indicating formation of 0.063 mM of [Ru(bpy)3]3+ and thus a yield of 0.65% for

photoinduced ET. The bleaching observed at 610 nm at long times (blue trace, DA = �0.0004) indicates reduction of

0.07 mM of T1 in good agreement with the quantity of oxidized chromophore. Used extinction coefficients: DεRu*450 =

εRu*450 – εRu2+450 = �11,300 M�1 cm�1 (Muller and Brettel, 2012); DεRu3+450 = εRu3+450 – εRu2+450 = �12,600 M�1 cm�1

(Kalyanasundaram, 1982b); εT1610 = 5600 M�1 cm�1 (Solomon et al., 2004). Small contributions to absorption changes at

450 nm due to T1 reduction and at 610 nm due to Ru3+ were neglected. The dashed pink line is the scaled kinetics of

emission at 610 nm, indicating that the 610 nm absorption transient contains contribution from other species than the

excited state. Green dashed line: difference between the dashed pink line and the 610 nm emission kinetics representing

transient bleaching of T1 absorption. Bottom: residuals for best fit according to the reaction scheme described in the text

(Scheme 1) and in the supplemental information. Best fit traces are superimposed on the red and blue experimental traces

as black lines both in the main window and in the inset.

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Interestingly, a careful comparison of the kinetics of the emission transient at 610 nm, expected to repre-

sent the decay of the [Ru(bpy)3]2+* excited state, and the absorption transient at 610 nm reveals significant

differences in the kinetics (Figure 1, compare blue and dashed pink lines). It appears clearly that the tran-

sient absorption trace recorded at 610 nm contains, in addition to the positive absorption attributable to

the [Ru(bpy)3]2+* excited state that has a small absorption at this wavelength (Yoshimura et al., 1993), a tran-

sient bleaching that can be related to a loss of Cu2+ absorption of the T1 site. The transient bleaching at

610 nm (green dashed line in Figure 1) is well described by two exponential functions for its formation

and decay with rate constants of 107 s�1 and 2 3 106 s�1, respectively. Considering a concentration of lac-

case of 37 mM, the observed forward rate is too fast to be compatible with a diffusion-limited reaction

between the sensitizer and the laccase. This fact leads us to postulate the formation of a relatively strong

association complex between the [Ru(bpy)3]2+ chromophore and LAC3 (Ka = 4.75 mM�1; determined by

kinetic simulations described below), present in equilibrium with the separated species before and directly

after the excitation flash. Such association complexes between ruthenium polypyridine and other transition

metal complexes with blue copper proteins have been reported before (Brunschwig et al., 1985; Goldberg

and Pecht, 1976). This interaction is usually attributed to the presence of negatively charged regions on the

protein surface interacting with the positively charged metal complexes. The crystal structure of a laccase

from Trametes versicolor revealed a dominance of negative charges of the protein and the substrate bind-

ing site as a small negatively charged cavity near copper T1 (Piontek et al., 2002). However, interactions

other than electrostatic may occur (Kurzeev et al., 2009). For LAC3, molecular docking calculations predict

a proximal binding position for [Ru(bpy)3]2+ at a distance as short as 11.2 A from the Cu T1 (Robert et al.,

2017). We note that the association constant Ka = 4.75 mM�1 implies that under the conditions of the exper-

iment shown in Figure 1, 12.5% of the [Ru(bpy)3]2+ complexes (1.9 mMof 15 mMemployed) are in association

with a LAC3 protein.

iScience 24, 102378, April 23, 2021 3

Scheme 1. Interactions between the [Ru(bpy)3]2+ photosensitizer and the LAC3 enzyme as identified by global

analysis of laser flash photolysis experiments

Reactions leading to formation of reduced laccase (LAC3-) and oxidized photosensitizer (Ru3+) are indicated by red

arrows. In the bimolecular pathway (left) Forster resonance energy transfer quenching of the [Ru(bpy)3]2+* excited state

largely outcompetes ET to the T1 CuII center in LAC3. In the association complex (right) charge recombination is about

10 times faster than dissociation leading to a low yield for formation of the target state Ru3+ LAC3– (red). Parameter values

were deduced from analysis of the data of Figure 1 (pH 6).

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The question that arises then is whether the observed bleaching of T1 absorption in the association complex

should be attributed to an electron or energy transfer process from the [Ru(bpy)3]2+ excited state to the copper

T1.We exclude formation of the doublet excited state of the T1 copper ion, as this statewas shown to decay to

the ground state in less than 1 ps, which would certainly not allow us to observe a bleaching of T1 absorption

persisting for hundreds of ns (Delfino et al., 2015). Formation of another,more long-lived excited state of the T1

Cu ion via Forster resonance energy transfer or via simultaneous exchange of an electron between T1 and the

excited [Ru(bpy)3]2+ complex (Dextermechanism) could explain the transient bleachingof T1 absorption.How-

ever, both energy transfer processes should lead to a concomitant recovery of the [Ru(bpy)3]2+ ground state for

which no indication is detectable in the transient absorption trace recorded at 450 nm. For this reason and to

account for the formation of stable Ru3+ and reduced T1 (Figure 1) we attribute the observed transient bleach-

ing of T1 to a photoinduced electron transfer reaction according to (Equation A):

�Ru2+ : T1/kcs

Ru3+ : T1-- /kbr

Ru2+ : T1 (Equation A)

where the rate constant kcs describes charge separation from the excited state of the chromophore yielding the

oxidized state of the chromophore (Ru3+) and reduced laccase (T1–), and kbr describes charge recombination

within the complex. It should be noted that the transiently formed species Ru3+ is characterized by an extinction

coefficient close to the one of the *Ru2+ excited state (see legend of Figure 1) that leads to only very weak mod-

ifications of the absorption at 450 nm. The dataset of Figure 1 was analyzed by a global kinetic simulation based

on the complete reaction scheme (Scheme 1, see also transparent methods section in the supplemental infor-

mation), and an excellent fit to the data was obtained (solid black traces and residuals in Figure 1) with all rate

constants well defined. From the kon and koff values describing formation of the association complex at two

different concentrations of LAC3an association constant ofKa = 4.75mM�1 wasdetermined together with values

for the rate constants kcs and kbr, of 8.53 106 s�1 and 1.83 106 s�1, respectively. Considering the large difference

in driving forces for the charge separation and charge recombination reactions (–DGcs = 1.52 eV versus –DGbr =

0.58 eV), the rate constants are surprisingly close. To obtain more information on the driving force dependance

of these ET rates the same process was investigated using [Ru(bpz)3]2+ (bpz: bipyrazine), a ruthenium sensitizer

with significantly higher Ru3+/Ru2+* and Ru3+/Ru2+ potentials (0.13 V and +2.23 V for [Ru(bpz)3]2+ compared with

�0.84 V and+1.26 V/NHE for [Ru(bpy)3]2+ (Kalyanasundaram, 1982b;Wehlin et al., 2017). Consequently, with the

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[Ru(bpz)3]2+ sensitizer the driving force for charge separation is about 1 eV lower than with [Ru(bpy)3]

2+, whereas

the driving force for charge recombination is increasedby the sameamount. Global kinetic simulation (Figure S3)

revealed an affinity of [Ru(bpz)3]2+ for LAC3 about 2 times lower than in the case of [Ru(bpy)3]

2+ (Ka = 2.36 mM�1

versus 4.75mM�1), implying a lower steady-state concentrationof complexes in the case of [Ru(bpz)3]2+. The rate

constants for the two ET reactions in the association complex between [Ru(bpz)3]2+ and LAC3 were determined

as kcs = 6.6 3 106 s�1and kbr = 1.2 3 106 s�1, close to the values found in the case of [Ru(bpy)3]2+.

With the known driving forces for the two reactions in eq (A) for the two sensitizers, the rates could be

analyzed in the framework of Marcus theory permitting an estimation of the reorganization energy and

distances for the ET reactions (Figure S4; see also transparent methods Section in the supplemental infor-

mation). A value of 1.05 G 0.07 eV for the reorganization energy of ET for both charge separation and

recombination can be deduced, which places the two ET reactions with high driving force in the inverted

region of the Marcus curve (Figure S4). The value for the reorganization energy is consistent with values

reported in literature for oxidation or reduction of T1 copper sites (0.6–0.8 eV) and the low internal reorga-

nization energy of the Ru2+/Ru3+ (or Ru2+*/Ru3+) transition (Di Bilio et al., 1997; Farver et al., 2004; Wijma

et al., 2007; Mines et al., 1996). Also, the distances extracted from the Marcus analysis, r = 12.5–13.5 A,

compare reasonably well with the RuII-to-T1 CuII distance of 11.2 A found by non-covalent docking simu-

lations (Robert et al., 2017). Interestingly, when the rates for charge separation and recombination were

analyzed separately (red and blue fit curves in Figure S4) the donor-acceptor distance for charge recombi-

nation was found to be 1 A larger than the distance for charge separation. Clearly additional data points on

the Marcus plot would be desirable to draw definite conclusions, but it is tempting to attribute this differ-

ence to the location of the electron transferred upon charge separation on the peripheral bipyridine ligand

in the *Ru2+ excited state in contrast to the location of the positive charge receiving the electron during

back ET on the central metal ion Ru3+. The fact that all rates determined by our global simulation can be

consistently described by Marcus theory with meaningful parameters for distances and reorganization en-

ergy further supports the proposed mechanism of electron transfer described by eq. (A).

The flash-induced energy and electron transfer reactions occurring upon excitation of [Ru(bpy)3]2+ in a so-

lution containing LAC3 are summarized in Scheme 1 and values for the case of [Ru(bpz)3]2+ are shown in

Scheme S1 in the supplemental information. It can be concluded from this investigation that diffusion-

controlled electron transfer from [Ru(bpy)3]2+* to LAC3 is short circuited by Forster energy transfer because

the latter operates over longer distances and that only excitation of a sensitizer present in a Ru:LAC3 as-

sociation complex at the time of photon absorption leads to electron transfer to the enzyme. The right-

side mechanism in Scheme 1 further predicts that the yield of effective laccase reduction is given by the

competition between the dissociation of the [Ru(bpy)3]3+:LAC3– complex described by the rate koff and

charge recombination within the complex described by the rate kbr. The association constant for the charge

separated state can be determined from the value of koff resulting from the global fit (koff = 1.2 3 105 s�1),

yielding Kacs = 8.5 mM�1. The slightly increased affinity of the sensitizer for LAC3 in the charge separated

state (Kacs = 8.5 mM�1 versus Ka = 4.75 mM�1) can be attributed to an increased electrostatic interaction

between oxidized Ru3+ and reduced laccase. The ratio of koff/(koff + kbr) then gives a yield of 6% for the

effective formation of Ru3+ and LAC3– from the charge separated state. With only 12.5% of the ruthenium

sensitizers engaged in such a complex, the overall quantum yield of persistent, not only transient, laccase

reduction (and Ru3+ formation) drops to 0.75%. Put together, these different phenomena explain well the

observed low yield of photoreduction of laccase from the excited state of the ruthenium sensitizer.

Although the simple reaction sequence proposed to occur in the association complexes (right panel of

Scheme 1) describes well the spectroscopic data, it is surprising that no energy transfer reaction from

[Ru(bpy)3]2+* to LAC3 had to be considered in the analysis. In other words, it appears that in the complex

the rate of energy transfer is slower than the rate of ET. This situation is reminiscent of a recent observation

in a flexible BODIPY-C60 dyad where photoinduced ET occurs in the folded conformer (R = 8.8 A), whereas

energy transfer is observed in extended conformers (R = 17–20 A) (Tran et al., 2020). Similarly, in studies on

single-walled carbon nanotubes tethered with porphyrins, excited-state energy transfer was found to be

absent in the sample with a short tether (Li et al., 2004). Our results therefore seem not to be a unique

case where at close distance the rate of energy transfer appears much slower than expected from the clas-

sical Forster rate-distance dependence. Although the different distance dependences between Forster en-

ergy transfer and ET (R�6 versus exponential) is expected to lead to a crossing of the k(r) curves with the rate

of electron transfer becoming faster than the rate of energy transfer for short distances, in our case this

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crossing occurs at too short distance to provide a simple explanation for the dominance of ET over energy

transfer (Figure S5). It might be argued that Forster energy transfer theory is an approximation based on

point dipole-dipole interaction restricted to weak coupling, valid at distances greater than R0 (dashed

green line in Figure S5), and extrapolation to significantly shorter distances might have to be taken with

care (Braslavsky et al., 2008; F}orster, 1959). However, there exist a huge body of experimental data where

the theoretical predictions hold even for quit short distances (Stryer and Haugland, 1967). We therefore

favor another explication related to the spin multiplicity in our system with a *Ru2+ triplet state and a

doublet ground state of the copper T1. Although examples of triplet-singlet and triplet-doublet energy

transfer reactions have been described (Naqvi, 1981; Cravcenco et al., 2019), the selection rules for angular

momentum conservation might result in a strong decrease of the energy transfer rates. A particularly strik-

ing example is the study by McCusker and coworkers on a model system where energy transfer was occur-

ring or inhibited as a function of the spin state of the acceptor (Guo et al., 2011). In our system, even two

orders of magnitude decrease in energy transfer rate due to the doublet spin state of the T1 Cu(II) could

have little effect at long distances (zR0) where the rate of electron transfer is slow but could be crucial

at short distances where the rate of electron transfer is fast enough to compete (Figure S5). It would be

interesting to investigate on the predominance of energy versus electron transfer in covalently linked ruthe-

nium-laccase constructs where the distance can be varied in a controlled manner.

Considering the results described above it appears clearly that an efficient reduction of laccase cannot be ob-

tained directly from the excited state of the sensitizer. In the following we assessed the possibility to overcome

these limitations by usingmethyl viologen (MV2+) as an electron relay between [Ru(bpy)3]2+* and LAC3. Systems

combining [Ru(bpy)3]2+ and MV2+ are probably the most extensively studied photoredox systems (Bock et al.,

1974; Sun et al., 1994; Mandal and Hoffman, 1984; Wilson et al., 1998). Acting as an oxidative quencher to the

photosensitizer’s excited state, this well-known mediator is converted into a long-lived reduced species that

could deliver electrons extracted from the chromophore to the enzyme even when the latter is present at

low concentration. Such an approach has helped to convey electrons to redox enzymes such as hydrogenases

under anaerobic conditions (Noji et al., 2014; Honda et al., 2016; Okura et al., 1979).

Laccase photoreduction via MV,+

To act as mediator, the chemical species to be used must meet several requirements. Besides a suitable

redox potential, it should have good solubility in aqueous solution, the oxidized form (necessarily present

in mM concentration for good dynamic quenching of the photosensitizer’s excited state) should have little

absorption in the visible to avoid absorption of excitation light, and, most importantly, the reduced mole-

cule must be able to act as a reductant for the enzyme. In contrast to many other small redox molecules

investigated as mediator, MV2+ was found fit for this duty. It offers the additional advantage that the

characteristic absorption features of its reduced state at 395 nm and 605 nm permit an easy tracking of

its reduction and reoxidation. Under anaerobic conditions, the emission of [Ru(bpy)3]2+* was quenched

by the addition of 10 mM MV2+ with a shortening of the lifetime of the excited state from 600 ns to

110 ns. The classic oxidative quenching of the [Ru(bpy)3]2+* by MV2+ involves a diffusion-limited encounter

followed by an intermolecular electron transfer process (Kalyanasundaram, 1982c; Sun et al., 1994). The

charge separated state (CSS) [Ru(bpy)3]3+ - MV,+ formed by dissociation of the encounter complex in

competition with the back ET is described by a cage escape yield of about 20% (Sun et al., 1994). The addi-

tion of LAC3 (17 mM) to the [Ru(bpy)3]2+/MV2+ mixture did not result in a significant change of the

[Ru(bpy)3]2+* excited state lifetime. This experimental observation suggests that the dominating deactiva-

tion pathway of the excited triplet state [Ru(bpy)3]2+* in the presence of both MV2+ and LAC3 is an ET pro-

cess to MV2+, which is faster than energy transfer to the laccase because of the much higher concentration

of MV2+ compared with LAC3. Transient absorption spectra were recorded at different time delays after the

laser flash (Figure S6). At 1 ms after the laser pulse, a bleaching around 450 nm and the typical positive ab-

sorption features of the MV,+ radical (395 nm and 605 nm) were observed indicating the formation of about

1 mMof the [Ru(bpy)3]3+ - MV,+ CSS by the oxidative quenching mechanism discussed above. Spectral evo-

lution up to 500 ms shows that the signature of reducedmethyl viologen disappears completely followed by

a bleaching around 610 nm, which denotes the consumption of the MV,+ radical and the loss of oxidized T1

in LAC3. These findings can be assigned to the charge shift from MV,+ to the T1 locus of the laccase while

the bleaching due to the [Ru(bpy)3]3+ species partially persists (Figure S6, red trace).

The absorption transients at specific wavelengths (Figure 2) allowed us to follow the concentration of the

different intermediate species. In the [Ru(bpy)3]2+/MV2+/LAC3 system, excitation of [Ru(bpy)3]

2+ leads to

6 iScience 24, 102378, April 23, 2021

Figure 2. Laccase photoreduction via MV,+

Kinetic traces of a mixture of [Ru(bpy)3]2+ (25 mM) and MV2+ (10 mM) in B&R buffer, pH = 4, with different concentrations

(0 mM, 16 mM, 30 mM) of LAC3. Absorption transients were recorded at 610 nm (upper part) or 450 nm (lower part). Black

lines show the results of a global fit yielding values for the bimolecular rate constants for charge recombination between

MV,+ and Ru3+ (3.13 109 M�1s�1), electron transfer fromMV,+ to T1 (0.383 109 M�1s��1), and oxidation of reduced T1 by

Ru3+ (charge recombination, 0.46 3 109 M�1s�1). Performing the experiment at pH 6 gave comparable results.

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the generation of 7.8 mM [Ru(bpy)3]2+* triplet excited state as deduced from the maximum bleaching

observed at 450 nm immediately after the laser flash (see legend of Figure 1 for extinction coefficients

used). As described before, the excited [Ru(bpy)3]2+* transfers an electron to the MV2+ leading to the for-

mation of 1.52 mM [Ru(bpy)3]3+ detected by the absorption bleach at 450 nm after decay of the excited

state, and the same amount of MV,+ radical, observed by the absorption increase at 610 nm at early times

after the flash (εMV+,605 = 13700 M�1 cm�1) (Watanabe and Honda, 1982). From these numbers a quantum

yield of ca. 19.5% for formation of the MV,+ radical is estimated. This yield agrees with reported values

for the cage escape yield for reduced MV,+ from the encounter complex (Kalyanasundaram, 1982c; Sun

et al., 1994). In the absence of LAC3 the MV,+ radical disappears in a second-order reaction (k = 3.1 3

109 M�1 s�1) due to charge recombination with [Ru(bpy)3]3+ yielding mirror-like absorption transients at

450 and 610 nm (Figure 2 blue traces). In the presence of 16 mM LAC3 (red traces) the decay of MV,+ at

610 nm is strongly accelerated, and the absorption transient turns into a bleaching indicating loss of

oxidized T1 of the laccase. With higher concentration of LAC3 the decay of MV,+ is further accelerated (Fig-

ure 2, green traces). The recovery of [Ru(bpy)3]2+ (bleaching at 450 nm) on the other hand slows down in the

presence of laccase, and the lifetime of both species, reduced T1 and oxidized chromophore, extends into

thems time range. Using the extinction coefficients at 610 nm εT1610z 5600M�1 cm�1 and εRu3+610z 2000

M�1 cm�1 (Kalyanasundaram, 1982b), a concentration of 0.7 and 0.9 mM of reduced T1 was estimated to be

formed after the laser flash for the two laccase concentrations, which represents a yield of 46 and 59%

compared with the quantity of MV,+ produced. This clearly illustrates that MV,+ can reduce the laccase

(DG = �1.13 eV) with diffusion-limited kinetics (k2 = 3.8 3 108 M�1 s�1). Importantly, the quantum yield

of formation of [Ru(bpy)3]3+ and [CuI] depends only on the competition between the forward ET from

MV,+ to LAC3 and the charge recombination of the [Ru(bpy)3]3+ - MV,+ CSS. This competition is in favor

of laccase reduction even for low concentrations of LAC3 because the flash-induced concentration of

MV,+ (and [Ru(bpy)3]3+, around 1 mM) is much smaller than the LAC3 concentration. It is important to recall

that in this system, optimization of laccase reduction is equivalent to optimization of formation of the

oxidized form of the ruthenium chromophore. The energetics and kinetics of the reactions occurring in

the [Ru(bpy)3]2+/MV2+/LAC3 system are summarized in Scheme S2 in the supplemental information.

Laccase photoreduction under continuous illumination in absence of O2

Time-resolved laser flashphotolysis studies such as thosepresented above are an important tool to investigate

the sequence of light-induced reaction steps and determine the relevant rate constants. On the other hand,

iScience 24, 102378, April 23, 2021 7

Figure 3. Laccase photoreduction under continuous illumination in the absence of O2

(A) Evolution upon illumination of absorption at 610 nm as a function of MV2+ concentration. [LAC3] = [Ru] = 30 mM,

[EDTA] = 10$mM, [MV2+] = 0–300$mMas indicated in Britton Robinson buffer pH 4.0 (25�C) in the absence of O2. Solid lines

show the best fit of a global simulation of the absorption kinetics with the reaction model described in the supplemental

information, transparent methods Section). Example of the evolution of concentrations of the different intermediates

during photoreduction of laccase as deduced from the global fit procedure for a concentration of MV2+ of 50 mM

(corresponding to the blue curve in panel A, reproduced as dotted black line). All red intermediates contribute to the

absorption at 610 nm, whereas the blue intermediates do not. Note that the last reduction step corresponds to a

transition T1redTNC2 / T1redTNC3 (compare dashed and solid blue lines between 230 and 420 s) meaning that T1 stays

almost fully reduced. This indicates that the third reduction of the TNC from T1red is endergonic (see text).

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photocatalytic processes employ continuous irradiationwhere reactions are governed by steady state concen-

trations significantlydifferent from thoseencountered in synchronized flashexcitation studies. To study the role

of theelectronmediatorbetween thechromophoreandLAC3 in thephotoreductionprocess under continuous

illumination, EDTA was added as an electron donor to the [Ru(bpy)3]2+/MV2+/LAC3 mixture. The [Ru(bpy)3]

3+

state is the only species able to oxidize EDTA, regenerating the light-active [Ru(bpy)3]2+ state that can engage

in subsequent ET reactions until the full four-electron reduction of laccase is achieved.

First, we studied LAC3 photoreduction in the absence of O2. In this context it is important to recall that un-

der anaerobic conditions the fully oxidized form of the enzyme corresponds to the so-called ‘‘resting

oxidized’’ (RO) form, which is structurally and functionally different from the fully oxidized native interme-

diate (NI) produced during catalytic turnover in the presence of O2. RO is proposed to evolve from NI by

protonation keeping all four Cu centers oxidized but ambiguity concerning the details of reduction of the

Cu centers in RO exists (Solomon et al., 2008; Jones and Solomon, 2015; Heppner et al., 2013, 2014).

A solution of sensitizer and LAC3 containing 10 mM EDTA and different concentrations of the MV2+ elec-

tron relay was irradiated with a white LED (filtered, 450<l < 700 nm) in the absence of O2, and the evolution

of absorption at 610 nm was followed as a function of illumination time (Figures 3A and S7). Before illumi-

nation, the absorption at 610 nm is exclusively attributable to the oxidized (CuII) T1 site. Upon illumination

this absorption decreases with a concave shape to reach zero, indicating complete reduction of the CuII T1

site. With increasing concentration of the electron mediator, MV2+, the time interval needed for T1 reduc-

tion is strongly decreased (Figure 3A). The plot of the reduction rate of T1 (defined as the reciprocal of the

time needed for complete reduction of T1) as a function of the concentration of MV2+ reveals a linear

dependence (Figure S8) with an acceleration by a factor of >10 for a MV2+ concentration of 300 mM

compared with the system without MV2+.The linear dependence can be traced back to the linear depen-

dence on MV2+ concentration of the quantum yield of MV2+ reduction prevailing at these low concentra-

tions (Figure S8). Rescaling of the absorption transients to match the time needed for disappearance of

T1 absorption reveals very similar shapes of the kinetics in the absence and presence of MV2+ (for all

concentrations studied, Figure S9), strongly suggesting that there is no fundamental difference in the

mechanism of reduction of the T1 site whether reduction occurs directly from the excited state of the chro-

mophore or via MV,+. After the absorption at 610 nm has decayed close to zero, indicating accumulation of

reduced T1, a re-increase of absorption at this wavelength is observed (Figure 3A) that can be clearly as-

signed to the accumulation of reduced methyl viologen radical MV,+ because its typical absorption

band at 395 nm grows concomitantly (Figure S7). Interestingly, especially at low concentrations of MV2+

8 iScience 24, 102378, April 23, 2021

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we noted a lag time between complete T1 reduction and the start of accumulation of MV,+ (Figures 3A and

S7 right). As observed for the reduction of T1, this time is shorter for higher concentrations of MV2+. It

seems reasonable to attribute this delay to the time needed for the total reduction of laccase (four reducing

equivalents). The fact that noMV,+ radical is detectable before complete laccase reduction implies that the

rate of generation of theMV,+ radical species by the excited sensitizer is slower than its consumption by the

laccase enzyme. To put numbers to this, at the highest MV2+ concentration a rate of formation of MV,+ of

2 mM/s is estimated. This means that with the employed concentration of LAC3 (30 mM) themaximum rate of

photoreduction of LAC3 is < 0.067 s�1.

To investigate the observed kinetics, we performed a global simulation of the absorption changes re-

corded with different concentrations of MV2+ (Figure 3). The presence of four different reduction states

of the laccase that can interact with two potential electron donors, [Ru(bpy)3]2+* or MV,+, makes the system

rather inimical to analyze and requires some simplifying assumptions. First, it seems sound to assume that

electron input into the LAC3 from either [Ru(bpy)3]2+* or MV,+ occurs via the T1 site (as supported by the

laser flash data, at least for the first reduction). Furthermore, we neglect the details of reactions occurring

within association complexes between [Ru(bpy)3]2+ and LAC3 and modeled laccase reduction from the

sensitizer’s excited state by a low-yield process. With these simplifications and because many rate con-

stants are reasonably well known from kinetic flash photolysis measurements and are independent of

MV2+ concentration, a reasonable fit to the experimental data could be obtained, which reproduces the

major features of the photoreduction assay with a common set of parameters (solid lines in Figure 3A).

With this model in hand, we can now investigate the critical parameters with the aim to extract information

on the mechanism governing the photoreduction of laccase and to determinate the rate constants for the

reactions describing internal electron transfer (IET) from T1 to the TNC center. In the following we will pre-

sent only the major results; details of the analysis are given in the transparent methods section in the sup-

plemental information.

We use the nomenclature of Sekretaryova et al. (Sekretaryova et al., 2019) to describe the redox states of

laccase from fully oxidized (T1oxTNC0) to the fully reduced form (T1redTNC3; with TNCx, x = number of elec-

trons on the TNC). The initial absorption decrease at 610 nm describes the loss of absorption from oxidized

T1 and corresponds to the consumption of three electrons, leading to reduced T1 and doubly reduced

TNC (T1redTNC2, dashed blue trace in Figure 3B). The first two IET steps from T1 to TNC, leading to the

formation of T1redTNC2, are fast compared with the photoproduction of MV,+, which is the rate limiting

step. Accordingly, the analysis was found to be not sensitive to the rates of forward electron transfer

from T1 to the TNC, provided they are faster than about 0.1 s�1. We assumed for the fits rate constants

of 1 s�1 as found in a pulse radiolysis study (Farver et al., 2011). The lag time following reduction of T1 cor-

responds to the uptake by the laccase of the fourth electron leading to full reduction of the enzyme

(T1redTNC3, solid blue line in Figure 3B). As can be seen in the profiles of the intermediates, this last reduc-

tion step corresponds to the transition T1redTNC2 / T1redTNC3 (compare dashed and solid blue lines be-

tween 230 and 420 s) meaning that T1 stays almost fully reduced during the process although T1 is the

entrance side for the electron. In fact, the kinetic parameters determined from the global fit clearly show

that the third reduction of the TNC from T1red is significantly endergonic (k+/k- z 1/4500; DG z +200

meV), in contrast to the first two IET steps that are close to equilibrium (Scheme 3). Because during catalytic

turnover all internal electron transfer steps leading to full reduction of the oxidized native intermediate (NI)

are fast, even in high-potential laccases as LAC3 used in our study,(Sekretaryova et al., 2019; Heppner et al.,

2014; Solomon et al., 1996), we attribute this sluggish third-electron reduction of the TNC to the fact that

under the anaerobic conditions of this experiment the oxidized enzyme is not in its native intermediate

form but in the resting oxidized (RO) state well described in the literature (Tollin et al., 1993). The charac-

teristics of internal electron transfer deduced from our experiment are in agreement with results from pulse

radiolysis and direct electrochemical measurements reporting a potential of the T2 Cu site in the RO state

of around 400 mV versus NHE, i.e. about 300 mV lower than the potentials of T1 and T3 (Farver et al., 2011;

Shleev et al., 2005). Although there is ‘‘insufficient driving force’’ for ET from reduced T1 to T2, our results

show that electron transfer occurs because the overall energetics is still favorable due to the low potential

of the MV,+ ‘‘substrate.’’ The possibility of uphill intramolecular electron transfer under certain conditions

had also been proposed based on electrochemical and quantum chemical studies (Shleev et al., 2012).

Furthermore, our conclusions fit with observations of Lazarides et al. on laccase photoreduction by a

porphyrin chromophore in presence of EDTA where it was found that the ESR signal of the T1 Cu(II) site

disappeared much faster (30 s) than the signal of the T2 Cu(II) species (30 min) (Lazarides et al., 2013). It

iScience 24, 102378, April 23, 2021 9

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appears that monitoring the accumulation of the reduced MV,+ radical is an indirect but valuable tool to

obtain information about IET events within the laccase enzyme, complementing measurements of absorp-

tion changes related to the oxidation state of T1, which are the only observables in the absence of MV2+.

Interestingly, the consistent analysis performed on this set of experiments, i.e., laccase photoreduction in

the absence and the presence of MV2+, reveals that in the absence of MV2+, where laccase reduction occurs

via the excited state of the chromophore, the enzyme is only reduced by three electrons (Figure S10). The

reason for this is the unfavorable pre-equilibrium resulting from the endergonic nature of the third reduc-

tion of the TNC, which makes the IET T1redTNC2 / T1oxTNC3 too slow to be compatible with the short

lifetime of the [Ru(bpy)3]2+* excited state. In contrast, the MV,+ species is long-lived and can progressively

reduce the state T1oxTNC3 present at low concentration in the above-mentioned equilibrium with

T1redTNC2 to form T1redTNC3. Of note, in the presence of dioxygen where all internal electron transfer

steps are nearly isoenergetic, the complete, four-electron reduction of laccase via [Ru(bpy)3]2+* is possible.

Finally, to obtain a satisfactory global fit of the absorption transients describing accumulation of MV,+ after

full, four-electron reduction of the enzyme (Figure 3A), we had to include in the simulation a feature that

accounts for a lack of the quantity of MV,+ accumulating (reaction A15 in the supplemental information,

transparent methods Section). The missing quantity, most clearly visible in the traces for low concentration

of MV2+ (see Figure S9), corresponds roughly to the quantity of enzyme present (30 mM). We therefore

tentatively attribute this feature to the formation of adducts of MV2+, with the enzyme being most efficient

when LAC3 becomes fully reduced. The ‘‘bound’’ MV2+ might still be reducible by [Ru(bpy)3]2+* but the

dissociation rate for MV,+ or Ru3+ from the Ru3+:MV,+:LAC3 complex might be too low to avoid charge

recombination between MV,+ and Ru3+.

Dioxygen consumption

With the finding that the presence of an electron mediator helps transferring electrons faster and more

efficiently to the LAC3, we then performed a set of dioxygen consumption experiments to characterize

the catalytic activity of the laccase when reduced by a sensitizer/electron mediator/electron donor

photoredox system. Under these aerobic conditions, O2 competes with the laccase for the electron

from the MV,+ species as it is well known that in aqueous solution MV,+ reacts rapidly with O2 to

form O2,– (or O2H

, at our operating pH) (Farrington et al., 1973). This competition between laccase

and O2 is clearly evident from transient absorption measurements revealing a markedly reduced effi-

ciency of flash-induced T1 reduction with increasing amounts of O2 (Figure S11). Surprisingly though,

when light-induced dioxygen consumption was measured with a Clark electrode, the effect of laccase

was much stronger than expected from this simple competition scheme. Figure 4 shows the rate of

O2 consumption as a function of LAC3 concentration in the absence and presence (250 mM) of MV2+.

In the absence of MV2+, the rate increases linearly with the concentration of LAC3, a behavior easily ex-

plained by an increased reduction of laccase from the excited state of the chromophore due to increased

formation of association complexes (see above). When MV2+ is present, but no laccase, a fast dioxygen

consumption rate is observed, which corresponds to the two-electron reduction of O2 to H2O2 via dis-

mutation of O2,– (eqs. B and D, Figure 5 red trace). Addition of LAC3 at a concentration much lower

than O2 (10–20 mM of LAC3 compared with 250 mM O2) leads to a very significant reduction of the dioxy-

gen consumption rate (Figure 4, black curve; Figure 5 blue trace).

MV,+ + O2 / MV2+ + O2,–

(B)

4 MV,+ + 4 CuII / 4 MV2+ + 4 CuI

(C)

2 O2,– + 2 H+ / O2 + H2O2

(D)

4 O2,– + 4 CuII / 4 O2 + 4 CuI

(E)

4 CuI + O2 + 4H+ / 4 CuII + 2 H2O

(F)

10 iScience 24, 102378, April 23, 2021

Figure 4. Rate of light-induced dioxygen consumption as a function of laccase concentration

[Ru] = 30$mM, [EDTA] = 10$mM, [MV2+] = 0 or 250$mM, [LAC3] = 0–81$mM in Britton Robinson buffer pH 4.0 (25�C). Initialdissolved oxygen concentration was 250 mM.

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2 MV,+ + O2 + 2 H+ / 2 MV2+ + H2O2 (B)+(D)

4 MV,+ + O2 + 4H+ / 4 MV2+ + 2 H2O (C)+(F) or 4x(B)+(E)+(F)

When laccase accepts electrons, a four-electron/four-proton total reduction of O2 is performed by the

enzyme (eqs. C and F) in addition to the two-electron/two-proton partial direct reduction of O2 to H2O2

(eqs. B and D). A substantial addition of an enzyme consuming half as much O2 for the same concentration

of photogenerated MV,+ could thus show up in the dioxygen consumption curve as a decrease in the di-

oxygen consumption rate compared with MV2+ alone. Therefore, the pattern in Figure 4 can be explained

by a competitive scheme where O2 is being progressively substituted by LAC3 as electron recipient from

MV,+ because the two paths distinctively produce end products, either H2O2 or H2O (Scheme 2). When

enough laccase is present to compete with O2 for electrons from MV,+ the rate increases and reaches a

saturation value given by the light intensity and efficiency of the sacrificial electron donor. However, the

effect of laccase on the rate of O2 consumption at laccase concentrations much lower than the concentra-

tion of dissolved O2 (250 mM) is surprisingly pronounced. This suggests that laccase reduction is more effi-

cient than expected from a simple competition between laccase and O2 for electrons from MV,+ as

observed in Figure S11. We therefore propose that laccase can be reduced not only by MV,+ but also

and quite significantly by the O2,– formed via electron transfer from MV,+ to O2 (eq. 4xB, E). Analysis of

the O2 consumption kinetics in the absence and presence of laccase (Figures 5 and S12) supports the inter-

pretation that it is indeed O2,– that is the dominant reductant for the laccase under these conditions. Lac-

case reduction by O2,– had also been reported based on pulse-radiolysis studies (Guissani et al., 1982;

(Pecht et al., 1977)). The two processes (eqs. (C)+(F) or 4x(B)+(E)+(F)) should then significantly prevent an

accumulation of H2O2. As a matter of fact, H2O2 concentration dropped dramatically as laccase was intro-

duced in dioxygen consumption experiments (Figure S14). The reaction sequence for photoreduction of

laccase under aerobic conditions is summarized in Scheme 2.

However, if O2,– can replace MV,+ for reduction of LAC3 as suggested by the O2 consumption experi-

ments, then the question arises why is the bleaching of T1 absorption, as detected in the laser flash

experiment (Figure S11), strongly reduced in the presence of O2? To answer this question, we performed

a global kinetic analysis of the oxygen consumption profile in the absence and presence of laccase (Fig-

ure 5). It is interesting to note that a first analysis of the O2 consumption kinetics (see Figure S12 and

transparent methods Section in the supplemental information) supports a slow reaction of superoxide

with LAC3 with a rate not exceeding 5 3 106 M�1s�1 (yielding a time constant of >1 ms for a concentra-

tion of O2 of 250 mM), about two orders of magnitude slower than the rate of laccase reduction by MV+,

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Figure 5. Light-induced oxygen consumption profile of a solution of 30 mM [Ru(bpy)3]2+, 1 mM EDTA, and 1 mM

MV2+ in B&R buffer pH 6, in the absence (red trace) and presence (blue trace) of 16 mM LAC3

Best fits (black solid lines) superimposed with the experimental data. The fit was performed according to a reaction

scheme considering adduct formation between O2,� and LAC3 followed by slow internal ET (for fit parameters see

supplemental information, transparent methods Section). Dashed blue line: simulation of O2 consumption in presence of

LAC3 but without considering reduction of LAC3 by O2,� (kD9-D12 = 0) keeping all other parameters constant. Dashed

green line: simulation of O2 consumption in the presence of LAC3 but without considering reduction of LAC3 by MV,+

(kD5-D8 = 0) keeping all other parameters constant. Comparing the dashed lines with the solid blue line indicates that

O2,�, and not MV,+, is the predominant reductant for the laccase under these conditions.

See Figures S12 and S13 in the supplemental information for further discussion.

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(see legend of Figure 2 and Scheme S2 in the supplemental information). If this applies also for the flash-

induced process, T1 reduction would be too slow to be detected on the timescale of the transient ab-

sorption measurement (1 ms) but could be still fast enough to be operational under the continuous light

conditions of the dioxygen consumption assay. Such a slow reduction of T1 by O2,– has been described

in pulse radiolysis studies where laccase was shown to be an efficient radical scavenger (Guissani et al.,

1982; Goldberg and Pecht, 1978). In these studies, O2,–, as other reducing radicals, was found to interact

with the laccase not through a direct reduction of the T1 center but instead by fast formation of a tran-

sient adduct with the protein followed by slow intra- or intermolecular electron transfer from the adduct

site to the T1. A refined kinetic simulation shows that such a scenario is fully compatible with the kinetics

of O2 consumption shown in Figure 5, provided that the adduct formation is reversible (see transparent

methods Section in the supplemental information). This scenario can account for the two orders of

magnitude difference in the apparent bimolecular rate constants for laccase reduction via MV,+ or

O2,– in a more convincing way because such a difference in bimolecular interaction rates would be diffi-

cult to rationalize. Unfortunately, neither the exact locus of the O2,– adduct nor a definite mechanism for

the intramolecular electron transfer have been determined in these pulse radiolysis investigations.

Although not favored by the authors of these earlier studies, it is tempting to hypothesize on the pos-

sibility of O2,– interacting with the enzyme at the level of the TNC considering the fact that this is the

natural binding site for O2 in laccases. The data presented here do not allow to draw conclusions on

this topic, but they might give indications for experiments dedicated to address these questions. Impor-

tantly though, our results clearly evidence the specificity of the enzyme acting as a sink for photogener-

ated electrons that it can efficiently recover either from an electron mediator or from a superoxide radical

and use them for the reduction of O2 to H2O. To visualize the action of laccase Figure S13 shows the

evolution of the concentration of key species during the photocatalytic cycle.

12 iScience 24, 102378, April 23, 2021

Scheme 2. Dioxygen reduction routes in the absence (2e�/O2, red)

or presence (4e�/O2, blue) of laccase

Thickness of arrows indicates predominance of pathways. The data on

the effect of LAC3 on O2 consumption rate in Figure 4 (pH 4) and

Figure 5 (pH 6) both demonstrate the participation of the enzyme,

suggesting that even at pH 4 O2,– is sufficiently long lived to reduce

laccase.

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Conclusion

Using synthetic chromophores to initiate light-driven electron transfer processes for photo-biocatalysis

can provide unique solutions to perform green chemical transformations. However, an innate limitation

in elaborating such systems resides in the presence of unproductive energy transfer processes that can

outcompete ET processes (Winkler, 2013). In this study, we clearly show that detrimental energy transfer

can be short circuited by the addition of a reversible electron acceptor, which transfers electrons from

[Ru(bpy)3]2+* to a laccase with high quantum yields. Even when the electron relay is reducing enough

to interact with O2 to form superoxide radicals, the laccase enzyme can efficiently accept electrons

from the latter species to catalyze O2 reduction and thereby short circuit the formation of H2O2. In

this way O2 functions as both, an electron shuttle to and oxidizing substrate for the laccase, leading

to an efficient photocatalytic cycle. With the presented thorough description of the charge transfer

and dioxygen reduction steps involving this MCO, we show that the dioxygen from air, usually carefully

eliminated from reaction media in synthetic systems mimicking photosynthesis, can become a key

element in photo-catalysis as a safe, renewable, and inexpensive alternative to sacrificial electron accep-

tors. In parallel to the consumption of O2 in such systems, a powerful oxidant, [Ru(bpy)3]3+ (1300 mV

versus NHE), is photo produced, ready to drive the oxidation of more tenacious organic substrates

(Scheme 3). Among future challenges ahead of us lie the use of the oxidative power of the oxidized chro-

mophore for further chemical transformations in lieu of the sacrificial electron donor used in this study.

Work along these lines is underway in our labs. In a broader context, this study also highlights the po-

tential of light-controlled electron delivery to enzymes as an interesting alternative to stopped-flow tech-

niques to reveal functional details of internal charge transfer and charge accumulation at the catalytic

sites. The interested reader is referred to Figure S15 in the transparent methods section of the supple-

mental information for some illustration.

Limitations of the study

This work provides insight on coupling of laccase with a photoredox system to create highly oxidizing spe-

cies using dioxygen as terminal electron acceptor. As the next step we are working on the application of

this system for oxidative catalysis by replacing the sacrificial electron donor EDTA used in this study by a

catalytic system for oxygen atom transfer reactions. Further work is required to analyze the effect of pH

on both the efficiency of laccase reduction and the oxidation catalytic cycle.

Resource availability

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead con-

tact, Winfried Leibl (winfried.leibl@cea.fr).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The published article includes all datasets/code generated or analyzed during this study.

iScience 24, 102378, April 23, 2021 13

Scheme 3. Energetic landscape of chromophore/electron relay/LAC3 system

Light absorption produces the strong oxidant [Ru(bpy)3]3+ stabilized by irreversible transfer of the electron via MV,+,

O2,–, and the laccase enzyme to O2. The dashed level indicates the lower potential of the TNC under anaerobic

conditions.

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METHODS

All methods can be found in the accompanying transparent methods supplemental file.

SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.isci.2021.102378.

ACKNOWLEDGMENTS

This work was supported by ANR Multiplet (ANR-15- CE07-0021-01) and by the French Infrastructure for

Integrated Structural Biology (FRISBI) ANR-10-INSB-05-01 and Labex Charmmmat (ANR-11-LABX-0039).

We thank Yolande Charmasson, Elise Courvoisier-Dezord, and the platform AVB: Analyze & Valorisation

de la Biodiversite at AMU for technical support in the production of the recombinant enzyme. WL would

like to thank Kenneth A. Johnson from KinTek Corp for making available the Global Kinetic Explorer soft-

ware. Without it, the kind of analyses presented in this work would not have been possible.

AUTHORS CONTRIBUTIONS

Conceptualization, A.A., J.S., T.T., C.H., and W.L.; Methodology, A.A., J.S., W.L., and T.T.; Investigation,

R.F., Y.M., N.T., A.Q., J.S., T.T., and W.L.; Formal Analysis, R.F., A.Q., Y.M., and W.L.; Resources, P.R.;

Writing—Original Draft, R.F. and A.Q., Writing—Review and Editing, M.S., J.S., A.A., T.T., F.B., C.H.,

and W.L.; Visualization, T.T., Y.M., and W.L.; Supervision, T.T., J.S., and W.L.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: January 29, 2021

Revised: March 11, 2021

Accepted: March 26, 2021

Published: April 23, 2021

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iScience, Volume 24

Supplemental information

Tracking light-induced electron transfer

toward O2 in a hybrid

photoredox-laccase system

Rajaa Farran, Yasmina Mekmouche, Nhat Tam Vo, Christian Herrero, AnnamariaQuaranta, Marie Sircoglou, Frédéric Banse, Pierre Rousselot-Pailley, A. JalilaSimaan, Ally Aukauloo, Thierry Tron, and Winfried Leibl

Supplemental Figures

Figure S1. Absorption spectra of samples recorded in B&R buffer, pH = 6. Concentrations: [Ru(bpy)3]2+ (17 µM), LAC3 (17 µM),

MV2+ (10 mM). The dashed line shows the emission spectrum of [Ru(bpy)3]2+*. Related to Fig. 1, Fig. 2, and Fig. 3.

Figure S2. Quenching of [Ru(bpy)3]2+* emission by laccase. Left: Emission decay at 610 nm for a solution of [Ru(bpy)3]

2+ (15 µM)

and different concentrations of LAC3 in B&R buffer, pH = 6 after excitation at 455 nm. Right: Stern-Volmer plot derived from

monoexponential fits to the data. The decrease in amplitude of the emission in presence of laccase is mainly attributable to an

inner filter effect caused by the absorption of the oxidized laccase overlapping the spectral region of [Ru(bpy)3]2+ emission (see

Fig. S1). For the highest LAC3 concentration part of decrease in initial emission amplitude is due to a dilution effect. Related to

Fig. 1 and Fig. S1.

Figure S3. Transient absorption kinetics of a solution of [Ru(bpz)3]2+ (15 µM) and LAC3 (36 µM) in B&R buffer, pH = 5.7 after laser

flash excitation at 455 nm. Inset: zoom of the data at long times. Bottom: residuals for best fit according to the reaction scheme

described in Scheme S1 below. The dashed pink line is the scaled kinetics of emission at 610 nm indicating that the 610 nm

transient contains contribution from other species than the excited state. Related to Fig. 1 and Scheme S1.

Figure S4. Plot of the first order rate constants of electron transfer in sensitizer-LAC3 complexes (eq. (A). Charge separation from

the excited state (red, kcs) and charge recombination to the oxidized sensitizer (blue, kbr). Sensitizers [Ru(bpy)3]2+ (open circles)

or [Ru(bpz)3]2+ (filled triangles). Solid lines show best fits to the Marcus equation (see section on calculations below) yielding 𝜆cs =

1.12 eV, 𝜆cr = 0.97 eV, rcs = 12.5 Å, and rcr = 13.5 Å as values for the reorganization energy and donor-acceptor distances for

charge separation and recombination. Related to Fig. 1, Fig. S3, Scheme 1, and Scheme S1.

Scheme S1. Interactions between the [Ru(bpz)3]2+ photosensitizer and the LAC3 enzyme identified by laser flash

photolysis experiments. Reactions leading to formation of reduced laccase (LAC3-) and oxidized photosensitizer (Ru3+)

are indicated by red arrows. In the bimolecular pathway (left) Förster resonance energy transfer quenching of the

[Ru(bpz)3]2+* excited state largely outcompetes with electron transfer to the T1 CuII center in LAC3. In the association

complex (right) charge recombination is about 10 times faster than dissociation leading to a low yield for formation of the

target state Ru3+ LAC3– (red). Parameter values were deduced from analysis of the data of Fig. S3 (pH 5.7). Related to

Fig. 1, Fig. S3, and Scheme 1.

Figure S5. Rates for Förster energy transfer (blue, Eq. 1) and electron transfer (red, Eq. 4) as a function of donor-acceptor distance

r. For the electron transfer rate, values for charge separation (-𝛥G = 1.52 eV and 𝜆 = 1.12 eV) from Fig. S4 were used and the

experimental rates for ET between the two chromophores and LAC3 are indicated by dots. The black dashed line represents the

maximal electron transfer rate (-𝛥G = 𝜆). The dashed blue line shows energy transfer rates 100 times slower than the Förster rates

to account for a hypothetical effect of selection rules related to the triplet/doublet spin multiplicity (see text). The value of the

Förster radius R0 = 26.4 Å is represented by the dashed green line. See Methods Section for equations and details.

At the employed concentrations of LAC3 the mean distance between the photosensitizers and LAC3 are larger than 30 Å. If the

position of LAC3 is taken at r = 0 Å a photosensitizer approaching (from the left side of the graph) will have to cross a k(r) curve

providing a rate faster than the diffusion limit for an interaction to occur. This occurs at about 23 Å in the case of “slow” Förster

energy transfer and at much larger distance for normal Förster energy transfer. At these large distances Marcus ET is many orders

of magnitude slower and therefore cannot occur. Related to Fig. 1, Scheme 1, Fig. S3, and Scheme S1.

Figure S6. Transient absorption spectra of a solution of [Ru(bpy)3]2+ (24 µM), MV2+ (10 mM) and LAC3 (30 µM) in B&R buffer, pH

=4 recorded at different delay times after laser flash excitation at 460 nm. Related to Fig. 2.

Scheme S2. Reaction scheme displaying energetic and kinetic characteristics after laser flash excitation of the

trimolecular [Ru(bpy)3]2+ / MV2+ / LAC3 system. At high concentrations of MV2+ compared to LAC3 (10 mM vs 30 µM) the

redox reaction between the [Ru(bpy)3]2+* excited state and MV2+ outcompetes energy transfer quenching to the T1 CuII

center in LAC3. The yield of formation of [Ru(bpy)3]3+ and T1 [CuI] then only depends on the competition between the

forward electron transfer from MV•+ to LAC3 and the charge recombination of the [Ru(bpy)3]3+ MV+• CSS, which clearly

favors the LAC3 route even for low concentrations of LAC3. The total quantum efficiency, limited by the escape yield of

MV2+ for formation of the charge-separated state, could be improved using a bichromophoric sensitizer (Wilson et al.,

1998). Related to Fig. 1, Fig. 2, Fig. S2, and Fig. S3.

Figure S7. Evolution of absorption spectra during continuous illumination of [Ru(bpy)3]2+ (30 µM), LAC3 (30 µM), MV2+ (150 µM),

and EDTA (10 mM) under anaerobic conditions. Left, Inset: absorption spectra at the indicated delay after onset of illumination.

The initial absorption spectrum (red) shows contributions from [Ru(bpy)3]2+ (𝜆=450 nm) and the CuII T1 site of laccase in its oxidized

form (𝜆=500-700 nm). After 1.6 min of illumination the 610 nm absorption band has disappeared leaving [Ru(bpy)3]2+ absorption

(𝜆=450 nm, blue). After a lag phase without spectral changes the typical spectrum of MV+• radical (395 nm, 605 nm) starts

growing (black). Right: Characteristics of the time dependence of absorption changes at 610 nm with a phase of CuII T1 reduction

(1) followed by a lag phase (2) and accumulation of MV+• radical (3). Related to Figure 3.

Figure S8. Effect of MV2+ concentration on the rate of photoreduction of T1 in a [Ru(bpy)3]2+/EDTA/MV2+ system. Rates k were

taken as the reciprocal of the time of illumination needed for the total bleach of the absorption at 610 nm. k0 is the rate in absence

of MV2+. Blue symbols indicate the simulated quantum yield of MV2+ reduction from the chromophore excited state which occurs

in competition with the intrinsic decay of the excited state, considering the escape yield for MV•+ of 20%. The good correlation

shows that laccase reduction is controlled by the yield of photoproduction of MV•+. Related to Figure 3A.

Figure S9. Laccase photoreduction under continuous illumination in absence of O2 for increasing concentrations of MV2+. Data of

Fig. 3A with rescaled time axis to match the time for complete reduction of T1 for any concentration of MV2+ with that in the

absence of MV2+ (for which complete reduction occurs at 620 s). The similar time courses of T1 reduction suggest the same

mechanism in the absence and in the presence of MV2+. The dash-dotted horizontal lines for the two lowest MV2+ concentrations

indicate the absorption expected after complete accumulation of reduced MV2+ which do not match with the final absorption of the

red and blue experimental traces. Related to Fig. 3.

Figure S10. Plot of the time course of oxidation of the sacrificial electron donor (EDTA) in the absence of MV2+ (blue curve) and

in the presence of 50 µM MV2+ (green curve). Reaction scheme and rate constants identical to those determined from the global

fit of absorption transients at 610 nm (Fig. 3A). Only three electrons per LAC3 are mobilized in the absence of the electron

mediator. In the presence of MV2+ three electrons are quickly consumed (in about 200 s), followed by a slower fourth reduction

and additional consumption of electrons due to accumulation of MV+•. The dashed lines show for the case of presence of 50 µM

MV2+ the partition of electron flux to laccase either via the excited state of the chromophore or via the electron mediator. For higher

concentrations of MV2+ ET from Ru* becomes negligible. Related to Fig. 3.

Figure S11. Transient absorption kinetics after laser flash excitation of [Ru(bpy)3]2+/[BV2+]/LAC3 in B&R buffer, pH 6 recorded at

610 nm under anaerobic conditions (blue) and in presence of increasing amounts of O2 (green, black, red). The decay indicates

kinetics of BV●+ oxidation, the extent of negative absorption at longer times indicates degree of laccase reduction (bleaching of T1

absorption). The experiment was performed using benzyl viologen (BV2+) instead of methyl viologen as electron relay. The less

negative redox potential of the BV2+/BV●+ couple compared to MV2+/MV●+ leads to a decrease of the second order rate constant

for electron transfer to O2 by a factor of 2. Analyses of the viologen radical decay kinetics in aerated aqueous solution yields

bimolecular rate constants for ET to O2 of 6.6·108 M-1s-1 and 3.3·108 M-1s-1 for MV2+ and BV2+, respectively. Related to Figure 2

and Scheme 2.

Figure S12. Light-induced oxygen consumption of a solution of 30 µM [Ru(bpy)3]2+, 1 mM EDTA and 1 mM MV2+ in B&R buffer

pH 6, in the absence and presence of 16 µM LAC3. Same data as Figure 5 of the main paper but best fit (black solid lines)

according to a simple reaction scheme considering parallel reduction of LAC3 by either O2●- or MV●+. (for fit parameters see Kinetic

Simulation section below). Dashed green line: simulation of O2 consumption in presence of LAC3 but without considering reduction

of LAC3 by MV●+ (kB5-B8 = 0) keeping all other parameters constant. Comparing the dashed lines with the solid blue line indicates

that O2●-, and not MV●+, is the predominant reductant for the laccase under these conditions.

In the absence of laccase, the constant slope is determined by the dismutation of superoxide which is faster than its production

(the steady state concentration of O2- is very low, see Fig. S13). In the presence of laccase, the initial O2 consumption rate is zero

because oxidation of O2- (regenerating O2, no net consumption) is faster than dismutation. In other words, laccase prevents

accumulation of sufficient concentration of O2- for dismutation to become kinetically competitive. Once LAC3 has recovered four

electrons, the enzyme starts to consume O2 by its normal catalytic activity. The action of O2- as a reversible electron relay between

MV●+ and LAC3 is not contributing to O2 consumption. Related to Fig. 5.

Figure S13. Visualization of the evolution of the concentration of key species during photocatalytic oxygen consumption

measurements in the absence (dashed lines) and in the presence of 16 µM LAC3. Dox is the concentration of oxidized electron

donor. In presence of LAC3 production of H2O2 is strongly reduced (yellow traces) and production of H2O is strongly accelerated.

In the absence of LAC3 O2●- quickly reaches a low stead-state concentration (0.6 µM) given by the ratio of the constant rate of

photoproduction via Ru* and MV●+ and the effective rate of dismutation which increases quadratically with [O2●-]. When LAC3 is

present O2●- level rises slowly because O2

●- is initially efficiently oxidized by LAC3 until a steady-state level is established which is

controlled by an ET step within the laccase which is rate limiting for O2 reduction catalysis by laccase. Related to Fig. 5, Fig. S12,

and Scheme 2.

Figure S14. Light dependent H2O2 production as a function of LAC concentration. Conditions: in 1 mL Britton and Robinson buffer

pH 4.0 (25°C) with [Ru]= 30 µM, [LAC3] = 0 to 90 µM, [EDTA] = 10 mM, and [MV2+] = 1.0 mM. Irradiation with a white Dolan-

Jenner mi-LED (250 mW.cm-2, 450<< 750 nm) until 100 % O2 consumed. Related to Fig. 5, Fig. S13, and Scheme 2.

Fig. S15. Simulation of a typical anaerobic photoreduction experiment to visualize the sensitivity of the absorption changes at 610

nm on rate constants for internal electron transfer from T1 to the TNC. Presence of excess MV2+ (2 mM) is assumed which permits

to neglect direct, inefficient interaction of the chromophore with laccase (reactions C1-C6 above). Light intensity was chosen to

produce 3 µM/s of MV●+ reductant. A) Variation of the rate of the first IET from T1 to TNC; B) Variation of the rate of the second

IET from T1 to TNC; C) Variation of the rate for reverse IET from the fully reduced TNC to T1. The latter corresponds to a change

from an exergonic to an endergonic reduction step for the storage of the third electron on the TNC. All forward rate constants

besides the ones varied are assumed not limiting (10 s-1). All backward rate constants besides the one varied in panel C are set

to 0. Related to Fig. 3.

Transparent Methods

Materials

Tris(2,2’-bipyridyl)dichlororuthenium(II) hexahydrate (99.5%), methyl viologen dichloride hydrate (98%)

and benzyl viologen dichloride (97%) were purchased from Sigma-Aldrich. The laccase LAC3 from

Trametes sp. C30 was produced as a recombinant enzyme in Aspergillus niger as described.(Klonowska

et al., 2005, Mekmouche et al., 2014)

Laser flash photolysis

For transient absorption and emission measurements an Edinburgh Instruments LP920 flash photolysis

spectrometer system was used with a Continuum Surelite OPO laser (5 ns pulse duration, typical energy

10 mJ) for excitation at 460 nm, a pulsed 450 W Xenon lamp for the probe light, and either a PMT

(Hamamatsu) or ICCD camera (Andor) as detectors. Presented data are typically averages over 10-20

measurements. Samples were prepared in sealed quartz cuvettes containing Britton–Robinson (B&R)

buffer at the indicated pH and were purged for 20 min with argon prior to each experiment.

Light-induced dioxygen consumption studies

Dioxygen consumption was measured by polarography using a model 781 oxygen meter (Strathkelvin

Instruments) or a Hansatech Oxygraph system with a micro-Clark electrode fitted to a temperature-

controlled glass chamber. Irradiation of the sample was performed through the glass chamber with a

Dolan JENNER LED (250 mW.cm-2) equipped with filters (450 nm <<700 nm). For the data in Fig. S11

irradiation was done with a Flexilux 600 Longlife 150 W 21 V halogen lamp through an ultraviolet cut-off

(λ≥375 nm) and a 23% neutral density filter.

H2O2 quantification

H2O2 production after illumination as a function of laccase concentration was determined by HRP assay.

40 µL of the reaction solution, filtered through a 3 kDa filter was added to a solution of 240 µL ABTS (2

mg/mL) in phosphate buffer pH 8.0 and 10 µL HRP (Sigma, 0.103 mg/mL) in 100 mM phosphate buffer

pH 8 was added. After 5 minutes of incubation the absorbance at 420 nm was recorded. Calibration was

done with reference measurements in the absence of light.

Kinetic spectroscopic studies

UV-vis absorption spectra were recorded on a Cary 50 spectrophotometer equipped with a homemade

irradiation setup including a Dolan JENNER LED (250 mW.cm-2) equipped with filters (450 nm <<700

nm). For each MV2+ concentration a quartz cuvette was filled with the reaction mixture and sealed in a

glovebox prior recording the corresponding spectrum.

Kinetic Simulation

Analysis of the kinetics of LAC3 photoreduction was performed using the KinTek Explorer simulation

program (Johnson et al., 2009b, Johnson et al., 2009a).

A) Simulation of flash-induced absorption changes in absence of MV2+ (Fig. 1, Fig. S3)

The simulation is based on the reaction scheme shown in Scheme 1:

A1) Rufree + Lox RuLox formation of association complex

A2) RuLox + light Ru*Lox excitation in association complex

A3) Ru*Lox RuLox intrinsic excited state decay in complex

A4) Ru*Lox RuIIILred electron transfer

A5) RuIIILred RuLox back electron transfer

A6) RuIIILred RuIII+ Lred dissociation of charge separated state

A7) Rufree + light Rufree* excitation of free chromophore

A8) Rufree* Rufree excited state decay of free chromophore

A9) Rufree* + Lox = Rufree + Lox excitation of free chromophore

The following rate constants were fixed: kA1= k-A6 = 1·109 M-1s-1; kA2= kA7 = 1·109 s-1; kA8 = 1.7·106 s-1.

Extinction coefficients are given in the legend of Fig. 1. In the simulation excitation was induced after

establishment of the association equilibria (reactions A1 and A6).

Best fit parameters: k-A1= 2.10.045·106 s-1; kA3= 1.540.06·106 s-1; kA4= 8.480.3·106 s-1; kA5=

1.80.025·106 s-1; kA6= 0.120.002·106 s-1; kA9= 7.40.12·106 s-1.

Association constants were determined as Ka = kon/koff = kA1/k-A1 and Kacs = k-A6/kA6.

B) Simulation of flash-induced absorption changes in presence of MV2+ (Fig. 2)

In presence of high concentrations of MV2+ ([MV2+] >> [Ru2+]) formation of association complexes

between Ru2+ and LAC3 can be neglected. The simulation is based on the following reaction scheme

starting with the flash-induced charge-separated state Ru3+ / MV●+ (initial concentrations 1.52 µM):

B1) RuIII + MV●+ Ru + MV2+ charge recombination

B2) MV●+ + Lox MV2+ + Lred LAC3 reduction

B3) RuIII + Lred Ru + Lox back electron transfer from reduced LAC3 to Ru3+

Best fit parameters: kB1= 3.10.01·109 M-1s-1; kB2 = 0.380.002·109 M-1s-1; kB3 = 0.460.004·109 M-1s-1.

When O2 was present (Fig. S11) the following reaction steps were added:

B4) MV●+ + O2 MV + O2●– oxidation of MV●+ radical by O2

B5) RuIII + O2●– Ru + O2 charge recombination between RuIII + O2

●–

With kB4= 0.7·109 M-1s-1; kB5 = 5·109 M-1s-1.

C) Simulation of anaerobic photoreduction of laccase (Fig. 3)

A simplified reaction scheme was used including the following reaction sequence:

C1) RuL + light RuL* excitation in association complex

C2) RuL* RuL intrinsic excited state decay

C3) RuL* + T1oxTNC0 RuIII + T1redTNC0

C4) RuL*+ T1oxTNC1 RuIII + T1redTNC1 effective reduction of laccase from

C5) RuL* + T1oxTNC2 RuIII+ T1redTNC2 the ruthenium excited state

C6) RuL* + T1oxTNC3 RuIII + T1redTNC3

C7) Ru III + D Ru + D+

C8) Rufree + light = Rufree* excitation of free chromophore

C9) Rufree* = Rufree intrinsic excited state decay

C10) Rufree*+ MV2+ RuIIIfree + MV●+ excitation and effective charge separation

C11) MV●+ + T1oxTNC0 MV2+ + T1redTNC0

C12) MV●+ + T1oxTNC1 MV2+ + T1redTNC1 Laccase reduction by MV●+

C13) MV●+ + T1oxTNC2 MV2+ + T1redTNC2

C14) MV●+ + T1oxTNC3 MV2+ + T1redTNC3

C15) MV2+ + T1redTNC3 MV2+:T1redTNC3 interaction of reduced MV with fully

reduced laccase

C16) RuIII + MV●+ RuII + MV2+ charge recombination

C17) T1redTNC0 T1oxTNC1

C18) T1redTNC1 T1oxTNC2 internal ET from T1 to TNC

C19) T1redTNC2 T1oxTNC3

C20) RuIIIfree + D Rufree + D+ oxidation of electron donor (EDTA)

C21) Rufree* + MV●+ Rufree + MV●+ quenching of ruthenium excited state by

energy transfer to MV●+

where:

- Ru, Ru*, and RuIII stand for the ground state, triplet excited state, and oxidized state of the

[Ru(bpy)3]2+ chromophore

- RuL is the fraction of chromophores in an association complex with laccase

- D and D+ are the reduced and oxidized states of the sacrificial electron donor (EDTA)

- MV2+ and MV●+ are the oxidized and reduced states of the methyl viologen electron relay

- T1 and TNC are the type 1 Cu ion site, and the trinuclear Cu catalytic center in the LAC3 enzyme

- The LAC3 enzyme is described by its redox state from fully oxidized (T1oxTNC0) to the fully

reduced form (T1redTNC3) with TNCx, x = number of electrons on the TNC

- Reactions 3-6: the successive reductions of T1 by the excited state of the sensitizer in the

preformed association complex

- Reactions 11-14: the successive reductions of T1 by MV●+

- Reaction 15 accounts for a hypothetical interaction of MV2+ with the fully reduced enzyme.

- Reactions 17-19: internal electron transfer reactions from T1 to the TNC in the laccase

- Reaction 21 accounts for energy transfer from Ru* to MV●+ having a broad absorption around

600 nm

Initial concentrations of chromophore-laccase association complexes (RuL) and free chromophore (Rufree)

were chosen according to the parameters determined before (Scheme 1). Other initial concentrations are

those used in the experiment. Reaction 10 takes into account the escape yield (Sun et al., 1994). For

computational reasons, effective electron transfer in association complexes between Ru and LAC3 were

simulated by apparent rates for electron transfer (reactions 3-6) in competition with the other deactivation

pathway for decay of the Ru excited state (reaction 2) and relative excitation rate (reaction 1). Potential

charge recombination between RuIIIfree and T1red was neglected. All reactions except intramolecular ET in

the laccase (17-19) and binding of MV2+ to fully reduced laccase (reaction 15) is considered irreversible.

For some reactions, rate constants are known from independent laser flash kinetic measurements.

Fixed rate constants used: intrinsic excited state decay of Ru*free: kC9= 1.6·106 s-1; oxidation of EDTA by

RuIII: kC7,C20 = 1.2·106 M-1s-1; energy transfer quenching of Ru*free by MV●+: kC21 = 8·109 M-1s-1. Forward

rate constants for IET were fixed to kC17-C19 = 1 s-1 (not limiting).

The essential result of the fit to the data is that, under the anaerobic conditions of this experiment where

the fully oxidized form of the laccase enzyme corresponds to the resting oxidized form (Solomon, 2016),

the third reduction of the TNC (reaction 19) is energetically uphill by about 200 meV. Values for rate

constants for internal electron transfer in the laccase derived from the simulation: k-C17=0.02 s-1,

k-C18=0.85 s-1, k-C19 ≈4500 s-1.

Other fitted rate constants: kC10 = 0.30.19·109 M-1s-1, kC16 = 0.70.6·109 M-1s-1. Large standard errors

like here indicate dependence between some of the parameters.

The traces in Fig. S10 were produced by plotting the flux integrals for reactions C7 and C20.

Simulations of a typical anaerobic photoreduction experiment to visualize the sensitivity of the absorption

changes at 610 nm on rate constants for internal electron transfer from T1 to the TNC (reactions C17-

C19) are shown in Fig. S15.

D) Simulation of O2 consumption (Fig. 5, Fig. S12, Fig. S13):

We consider the following simplified reaction scheme with photoproduction of MV●+ via the Ru excited

state as above, including charge recombination between RuIII and MV●+ and reduction of RuIII by the

sacrificial electron donor EDTA. As under aerobic conditions all internal electron transfer steps are fast,

we use the simplified representation of the five redox states of laccase L0 - L4 with L0 the fully oxidized

and L4 the fully (4 e-) reduced form. Protonation equilibrium of superoxide is assumed to be fast and is

not explicitly considered.

D1) Ru + light Ru* excitation and

D2) Ru*+ MV2+ RuIII + MV●+ effective charge separation

D3) RuIII + D RuII + D+ oxidation of electron donor

D4) RuIII + MV●+ RuII + MV2+ charge recombination

D5) MV●+ + L0 MV2+ + L1

D6) MV●+ + L1 MV2+ + L2 Laccase reduction by MV●+

D7) MV●+ + L2 MV2+ + L3

D8) MV●+ + L3 MV2+ + L4

D9) O2●– + L0 O2 + L1

D10) O2●– + L1 O2 + L2 Laccase reduction by O2

●–

D11) O2●– + L2 O2 + L3

D12) O2●– + L3 O2 + L4

D13) L4 + O2 L0 + 2 H2O 4-electron reduction of O2 by laccase

D14) MV●++ O2 MV2+ + O2●– O2 reduction by MV●+

D15) O2●– + O2

●– + 2 H+ H2O2 + O2 superoxide disproportionation

D16) H2O2 + H2O2 O2 + 2 H2O H2O2 disproportionation

Initial concentrations were: [light] = 1 µM (constant), [Ru] = 30 µM, [D] = 1 mM, [MV2+] = 1 mM, [L0] = 0,

16 µM, [O2] = 253 µM.

Fixed rate constants used: kD2 = 0.3·109 M-1s-1, kD3 = 1.28·106 M-1s-1, kD4 = 1.27·109 M-1s-1, kD5-D8 =

0.5·109 M-1s-1, kD14 = 0.67·109 M-1s-1, kD15 = 1·106 M-1s-1, kD16 = 17 M-1s-1.

Rate constants determined by global fit of the O2 consumption kinetics in Fig. 5:

kD1 = 0.0260.00002 s-1, kD9-D12 = 0.03-0.3·106 M-1s-1, kD13 = >103 M-1s-1,

Reaction 4 is essentially not operating due to the low steady-state concentrations of RuIII and MV●+. At

the high concentration of MV2+ used in the experiment laccase reduction directly from Ru* can be safely

neglected.

Simulation of O2 consumption with adduct (Fig. 5):

Same scheme as above with eqs D9-D12 replaced by:

D9a) O2●– + L0 O2

●–:L0

D9b) O2●–:L0 O2 + L1

D10a) O2●– + L1 O2

●–:L1

D10b) O2●–:L1 O2 + L2 Laccase : O2

●– adduct formation followed by

D11a) O2●– + L2 O2

●–:L2 laccase reduction

D11b) O2●–:L2 O2 + L3

D12a) O2●– + L3 O2

●–:L3

D12b) O2●–:L3 O2 + L4

Fixed rate constants used: kD9a-12a = 0.5·109 M-1s-1.

Rate constants determined by global fit of the O2 consumption kinetics in Fig. 5: k-D9a--D12a = 0.90.8·106

M-1s-1, kD9b = 2.60.6·106 s-1, kD10b = 0.380.05·106 s-1, kD11b = 0.080.08·106 s-1, kD12b = 0.230.5·106 s-1.

Other rate constants as before.

Calculation of energy and electron transfer rates

Förster-type resonance energy transfer:

To investigate the competition between energy transfer quenching and photo-induced electro transfer

quenching of the ruthenium excited state it is useful to simulate the distance-dependence of both

processes. The rate of oscillating dipole energy transfer between a donor and acceptor molecule can

be deduced from equation (1) proposed by Förster (Főrster, 1959, Lakowicz, 2006)

𝑘𝐸𝑛𝑇 (𝑟) =1

𝜏𝐷(

𝑅0

𝑟)

6

(1)

where τD the excited state lifetime of the donor in the absence of acceptor, R0 is the Förster distance at

which energy transfer is 50% efficient, and r is the distance between donor and acceptor. The coupling

between the dipolar transitions of donor and acceptor is determined by the Förster radius given by

𝑅06 =

9000∗ln(10)∗𝜅2∗𝛷𝐷∗𝐽

128∗𝜋5∗𝑛4∗𝑁𝐴 (2)

where κ2 is the dipole orientation factor, ΦD the quantum yield of the donor emission, n the refractive

index, NA Avogadro’s number, and J the spectral overlap integral

𝐽() = ∫ 𝐹𝐷() ∗ 𝜀𝐴() ∗ −4 𝑑∞

0 (3)

in units of mol-1 dm3 cm3 (Braslavsky et al., 2008), which contains the normalized emission spectrum of

the donor, FD, and the absorption spectrum of the acceptor, εA as a function of wavenumber, ν. J was

evaluated from the data in Fig. S1 to yield J = 7.4·10-14 mol-1 dm3 cm3. Using as values for the other

parameters in eq (2) κ2 = 0.476 (Steinberg, 1971), ΦD = 0.042 (Kalyanasundaram, 1982), and n = 1.4

Lakowicz (2006) R0 is determined to R0 = 26.4 Å. Together with the value τD = 588 ns from Fig. S2 this

allows to draw the kEnT(r) plot shown in Fig. S5.

Intra-complex electron transfer rate

Classical Marcus theory describes the rate of an exergonic reaction as (Marcus and Sutin, 1985, Millett

and Durham, 2002)

𝑘𝑒𝑡(𝜆, 𝛥𝐺°′, 𝑟) = 1 ∗ 1013 exp(−𝛽(𝑟 − 𝑟0)) ∗ exp (−(𝛥𝐺°′+𝜆)2

4𝜆𝑘𝐵 𝑇) (4)

Where 𝛥G°’ is the free energy of reaction, 𝜆 is the reorganization energy, r is the edge to edge distance

between the two redox centers, r0=3.6 Å is the van der Waals contact distance, and β =1.4 Å-1. (Millett

and Durham, 2002).

Supplemental References

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FŐRSTER, T. 1959. 10th Spiers Memorial Lecture. Transfer mechanisms of electronic excitation. Discussions of the Faraday Society, 27, 7-17.

JOHNSON, K. A., SIMPSON, Z. B. & BLOM, T. 2009a. FitSpace explorer: an algorithm to evaluate multidimensional parameter space in fitting kinetic data. Anal Biochem, 387, 30-41.

JOHNSON, K. A., SIMPSON, Z. B. & BLOM, T. 2009b. Global kinetic explorer: a new computer program for dynamic simulation and fitting of kinetic data. Anal Biochem, 387, 20-9.

KALYANASUNDARAM, K. 1982. Photophysics, photochemistry and solar energy conversion with tris(bipyridyl)ruthenium(II) and its analogues, Amsterdam.

KLONOWSKA, A., GAUDIN, C., ASSO, M., FOURNEL, A., RÉGLIER, M. & TRON, T. 2005. LAC3, a new low redox potential laccase from Trametes sp. strain C30 obtained as a recombinant protein in yeast. Enzyme and Microbial Technology, 36, 34-41.

LAKOWICZ, J. R. 2006. Energy Transfer. In: LAKOWICZ, J. R. (ed.) Principles of Fluorescence Spectroscopy. Boston, MA: Springer US.

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MEKMOUCHE, Y., ZHOU, S., CUSANO, A. M., RECORD, E., LOMASCOLO, A., ROBERT, V., SIMAAN, A. J., ROUSSELOT-PAILLEY, P., ULLAH, S., CHASPOUL, F. & TRON, T. 2014. Gram-scale production of

a basidiomycetous laccase in Aspergillus niger. Journal of Bioscience and Bioengineering, 117, 25-27.

MILLETT, F. & DURHAM, B. 2002. Design of photoactive ruthenium complexes to study interprotein electron transfer. Biochemistry, 41, 11315-24.

SOLOMON, E. I. 2016. Dioxygen Binding, Activation, and Reduction to H2O by Cu Enzymes. Inorg Chem, 55, 6364-75.

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SUN, H., YOSHIMURA, A. & HOFFMAN, M. Z. 1994. Oxidative Quenching of the Excited-State of Tris(2,2'-Bipyridine)Ruthenium(2+) Ion by Methylviologen - Variation of Solution Medium and Temperature. Journal of Physical Chemistry, 98, 5058-5064.

WILSON, G. J., LAUNIKONIS, A., SASSE, W. H. F. & MAU, A. W. H. 1998. Chromophore-Specific Quenching of Ruthenium Trisbipyridine−Arene Bichromophores by Methyl Viologen. The Journal of Physical Chemistry A, 102, 5150-5156.